Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: Effect of FHL3 in progression of pancreatic cancer.

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques:

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: FHL3 referred to PDAC progression. ( A and B ) IHC staining and statistical analysis of FHL3 for sections from 55 matched different lesion stages tissues of PDAC and para-tumor normal area, which showed the higher expression level of FHL3 in PDAC, more than 3-fold, as compared with normal tissue, p<0.001. ( C ) Survival analysis via Kaplan-Meier analysis in 55 PDAC samples which showed higher expression level of FHL3 was accompanied with worse prognosis, p=0.0168. ( D ) WB assay of 8 matched fresh frozen PDAC samples, which showed higher expression level of FHL3 in PDAC tissues more than 1.5-fold, p<0.01.

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: Immunohistochemistry, Expressing

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: Univariate and multivariate analysis of clinicopathological variables and FHL3 expression associated with overall survival

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: The expression level of FHL3 referred to EMT makers and metastasis in pancreatic cancer cells. ( A ) IF assay showed the expression level of FHL3 in four pancreatic cell lines and one normal pancreas cell line, and the expression rank was: PANC1>BXPC3>CFPAC1>MIA PACA2>HPDE. ( B and C ) WB assay showed the same results, and PANC1 and BXPC3 held the highest expression level of FHL3, which were more than 4-fold higher when compared to HPDE, p<0.001. ( D and E ) transwell assay for evaluating the migration ability in four pancreatic cell lines and one normal pancreas cell line, in which the migration rank was: PANC1>BXPC3>MIA PACA2>CFPAC1> HPDE. ( F ) linear relationship fitting analysis showed migration ability was correlate with the expression level of FHL3, r=0.8108, p=0.0371. TCGA data analysis showed FHL3 was negatively correlated to expression level of EMT maker E-cadherin ( G1 ) (r=-0.220, p=0.003), and passively correlated to EMT maker vimentin ( G2 )(r=0.627, p<0.001) and EMT associated transcription factors snail1 ( G3 ) (r=0.452, p<0.001, Fig.2G 3 ) and twist1 ( G4 ) (r=0.484, p<0.001).

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: Expressing, Transwell Assay, Migration

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: FHL3 knockdown restrained migration and reversed EMT phenotype in pancreatic cancer cells. ( A1 and B1 ) IF assay of FHL3 in PANC1_NC/KD 1-3 , BXPC3_NC/KD 1-3 , which showed KD1 sequence was the most efficient. (A2 and B2) WB assay of FHL3 in PANC1_NC/KD 1-3 and BXPC3_NC/KD 1-3 , which showed the expression level of FHL3 was downregulated more than 80% in PANC1_KD1 and BXPC3_KD1 cells, p<0.001. ( C ) 48h- transwell assay showed more than 40% decreasing of migration ability of PANC1_KD1 and BXPC3_KD1 cells, as compared with PANC1_NC and BXPC3_NC cells, P<0.01. ( D ) 72h-wound healing assay showed KD1 cell lines held more blank area as compared with NC cell lines, p<0.05. ( E1 ) WB assay showed FHL3 knockdown reversed the expression level of EMT associated proteins and transcriptional factors, except zeb1.

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: Knockdown, Migration, Sequencing, Expressing, Transwell Assay, Wound Healing Assay

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: FHL3 regulated EMT mainly by TGFβ1/Akt/GSK3β/ubiquitin pathway. ( A1 and A2 ) WB assay showed FHL3 knockdown made downregulation of TGFβ1, smad 2/3 , and smad 4 in PANC1_KD1 and BXPC3_KD1 in total protein level. ( B1 and B2 ) WB assay of nucleus-cytoplasm protein showed that FHL3 knockdown hardly changed the expression level of smad 2/3 and smad 4 in nucleus in PANC1_KD1 and BXPC3_KD1 cells. ( C1 and C2 ) In PANC1_KD1 and BXPC3_KD1 cells, FHL3 knockdown exactly downregulated the absolute and relative expression of phosphorylated AKT (ser473-AKT) more than 50%, p<0.01; and also downregulated the absolute and relative expression of phosphorylated GSK3β (ser9-GSK3β) more than 50%, p<0.01; and upregulated the absolute and relative expression of phosphorylated GSK3β (try216-GSK3β) more than 2-fold, p<0.001. ( D1 ) 0.25μM and 0.50μM GSK3β inhibitor almost eliminated the effect, promoting the TGFβ1/AKT/GSK3β/ubiquitin process, caused by FHL3 knockdown. As treated with GSK3β inhibitor, GSK3β (try216-GSK3β) and E-cadherin were downregulated, snail1 and twist1 were upregulated. ( D2 ) 0.25μM and 0.50μM GSK3β inhibitor reversed the migration ability of PANC1_KD1 and BXPC3_KD1 cells.

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: Ubiquitin Proteomics, Knockdown, Expressing, Migration

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: LIM-3 was the pivotal domain for FHL3 competitively binding to GSK3β. ( A1 and A2 ) Transfection with GSK3β, FHL3 (+:5ug, +++:15ug), snail1 and twist1 in HEK293T for 48h followed by CO-IP assay, which showed the higher FHL3 expression was accompanied with the higher expression of snail1 and twist1, p<0.01; meantime, the higher FHL3 expression made less snail1 and twist1 which binding with GSK3β, p<0.001. ( B ) Truncated forms form FHL3. ( C ) Transfection with GSK3β and truncated forms for 48h in HEK293T cells, and the CO-IP assay showed only truncated forms which containing LIM-3 domain could bind with GSK3β.

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: Binding Assay, Transfection, Co-Immunoprecipitation Assay, Expressing

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: FHL3 knockdown inhibited pancreatic tumor growth and metastasis in vivo. ( A ) experiments process in vivo. ( B ) tumor volume of PANC1_KD1 and PANC1_NC, 4 weeks after orthotopic transplantation; and the tumor volume of PANC1_KD1 group was about 1/3 of PANC1_NC group, p<0.01. ( C ) FHL3 knockdown made the weaker Ki67 staining in tumor sections of PANC1_KD1 group. ( D ) H&E staining of tumor section. ( E ) IHC staining of FHL3 of tumor section. ( F1a , F1b ) lung metastasis from circular pancreatic tumor cells, and FHL3 knockdown made the lower occurrent rate of lung metastasis in PANC1_KD1 and BXPC3_KD1 groups, p<0.05. ( F2a , F2b ) hepatic metastasis from orthotopic transplantation tumor, and FHL3 knockdown made the lower occurrent rate of hepatic metastasis in PANC1_KD1 and BXPC3_KD1 groups, p<0.05.

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: Knockdown, In Vivo, Transplantation Assay, Staining, Immunohistochemistry

Journal: Aging (Albany NY)

Article Title: FHL3 promotes pancreatic cancer invasion and metastasis through preventing the ubiquitination degradation of EMT associated transcription factors

doi: 10.18632/aging.102564

Figure Lengend Snippet: Mechanism of FHL3 in regulation of EMT process. Protein which involved in signaling: AKT (AKT, ser437-AKT), GSK3β (GSK3β, ser9-GSK3β, try216-GSK3β), FHL3, snail1, twist1, TGFβ1, smad (smad 2/3 , smad 4 ), and E-cadherin.

Article Snippet: What more, the work concentration of antibody against FHL3 (Proteintech, China) was 1:150, and 1:200 for Ki67 proliferation index (Abcam).

Techniques: